1. Field of the Invention
The present invention relates to novel parathyroid hormone peptide (PTH) derivatives. In particular, the invention relates to PTH derivatives having one or more amino acid substitutions that confer PTH-1/PTH-2 receptor agonist or antagonist properties to the derivatives.
2. Description of Related Art
Full understanding of the complex biological roles of parathyroid hormone (PTH) and efforts to utilize its therapeutic potential require dissection of the multiple signaling patterns and cellular pathways of action of the hormone. PTH binds and activates specific receptors in renal and osseous target cells that also recognize PTH-related peptide (PTHrP) (Kronenberg, H., et al., xe2x80x9cThe PTH/PTHrP receptor: one receptor for two ligands,xe2x80x9d in Genetics of Endocrine and Metabolic Disorders, Thakker, R., ed., Chapman and Hall, London (1997), pp. 389-420). In renal and osteoblastic cell lines, PTH triggers several parallel intracellular signaling responses, including activation of adenylyl cyclase (AC), protein kinase A (PKA), phospholipase C (PLC) and protein kinase C (PKC) and generation of second messengers such as cyclic AMP (cAMP), inositol trisphosphate (IP3), diacylglycerol and increased cytosolic free calcium (Cai++) (Abou-Samra, A. B., et al., Proc. Natl. Acad. Sci. U.S.A. 89(7):2732-2736 (1992); Azarani, A., et al., J. Biol. Chem. 271(25):14931-14936(1996); Bringhurst, F. R., et al., Endocrinology 132(5):2090-2098 (1993); Civitelli, R., et al., Am. J. Physiol. 255(5 Pt 1):E660-667 (1988); Donahue, H. J., et al., J. Biol. Chem. 263:13522-13527 (1988); Dunlay, R., and Hruska, K., Am. J. Physiol. 258(2 Pt 2):F223-231 (1990); Fujimori, A., et al., Endocrinology 128(6):3032-3039 (1991); Fujimori, A., et al., Endocrinology 130(1):29-36 (1992); Guo, J., et al., Endocrinology 136(9):3884-3891 (1995); Janulis, M., et al., Endocrinology 133:713-719 (1993); Jouishomme, H., et al., J. Bone Miner. Res. 9(6):943-949 (1994); Juppner, H., et al., Science 254(5034):1024-1026 (1991); Pines, M., et al., Bone 18(4):381-389 (1996); Seuwen, K., et al., Brit. J. Pharm. 114(8):1613-1620 (1995); Siegfried, G., et al., Endocrinology 136(3):1267-1275 (1995).
To date, two structurally related but distinct species of PTH receptors have been cloned (Abou-Samra, A. B., et al., Proc. Natl. Acad. Sci. U.S.A. 89(7):2732-2736 (1992); Usdin, T. B., et al., J. Biol. Chem. 270(26):15455-15458 (1995); Schipani, E., et al., Endocrinology-132(5):2157-2165 (1993)). The first of these, type A, was isolated from both bone and kidney cells and shown to transduce multiple signaling responses to PTH-(1-34) or PTHrP(1-36) when heterologously expressed in cells that lack endogenous type 1 PTH/PTHrP receptors (PTHRs) (Abou-Samra, A. B., et al., Proc. Natl. Acad. Sci. U.S.A. 89(7):2732-2736 (1992); Azarani, A., et al., J. Biol. Chem. 271(25):14931-14936 (1996); Bringhurst, F. R., et al., Endocrinology 132(5):2090-2098 (1993); Guo, J., et al., Endocrinology 136(9):3884-3891 (1995); Pines, M., et al., Bone 18(4):381-389 (1996); Jobert, A.-S., et al., Endocrinology 138(12):5282-5292 (1997); Schneider, H., et al., Eur. J. Pharm. 246(2):149-155 (1993)).
Previous efforts to define the contributions of specific regions of the PTH molecule to its binding and signaling properties have been undertaken mainly by use of complex in vivo bioassays, organ cultures, isolated cell membranes or cell lines, generally of rodent origin, that may express more than one type of endogenous PTH receptors (Janulis, M., et al., Endocrinology 133:713-719 (1993); Siegfried, G., et al., Endocrinology 136(3):1267-1275 (1995); Yamamoto, S., et al., Endocrinology 138:2066-2072 (1997); Jouishomme, H., et al., Endocrinology 130(1):53-60 (1992); Segre, G. V., et al., J. Biol. Chem. 254:6980-6986 (1979); Tregear, G. W., and Potts, J. T., Jr. Endocr. Res. Commun. 2:561-567 (1975); Takasu, H., et al., Endocrinology 137(12):5537-5543 (1996); Orloff, J. J., et al., Am. J. Physiol. 262(5 Pt 1):E599-607 (1992)). For example, relatively few modifications have been made to the extreme amino terminus of PTH: Synthetic [desamino-Ala-1]bPTH-(1-34) was disclosed by Tregear et al. (Endocr. Res. Commun. 2:561-570, page 566 (1975)), by Goltzmann et al. (J. Biol. Chem. 250:3199-3203, (1975)), and by Parsons et al. (Pharmacology of parathyroid hormone and some of its fragments and analogues. In: Talamage R V, Owen M, Parsons J. A. (Eds.) Calcium-Regulating Hormones. American Elsevier, New York, pp 33-39 (1975)); synthetic [desamino Ser1]-hPTH(1-34) was disclosed by Tregear et al. (Endocr. Res. Commun. 2:561-570 (1975)) and by Rixon et al. (J. Bone and Mineral Res. 9: 1179-1189 (1994)); synthetic 1-desamino-hPTH-(1-31) was disclosed by Rixon et al. (J. Bone and Mineral Res. 9: 1179-1189, (1994))and synthetic [Ala1]-hPTH(1-34), and [Gly1]-hPTH(1-34) were disclosed by Tregear et al. (Endocr. Res. Commun. 2:561-570, page 563 (1975)).
Early structure/function studies of bovine PTH-(1-34), performed with isolated renal membranes, identified the key role of the carboxyl(C)-terminal bPTH-(25-34) region for receptor binding and of the amino(N)-terminus (i.e., Ser1) for AC activation (Segre, G. V., et al., J. Biol. Chem. 254:6980-6986 (1979); Tregear, G. W., and Potts, J. T., Jr. Endocr. Res. Commun. 2:561-567 (1975)). Later work conducted in vitro with intact renal tubules or with cultured renal or bone cells, however, indicated that N-truncated analogs such as PTH-(3-34), although unable to stimulate AC, could fully activate PKC and could regulate certain PKC-dependent distal biologic responses (Janulis, M., et al., Endocrinology 133:713-719 (1993); Siegfried, G., et al., Endocrinology 136(3):1267-1275 (1995); Jouishomme, H., et al., Endocrinology 130(1):53-60 (1992)). Amino-truncated analogs of PTH-(1-34) also were found to increase PLC activity or Ca1++ in some cells (Donahue, H. J., et al., J. Biol. Chem. 263:13522-13527 (1988); Fujimori, A., et al., Endocrinology 128(6):3032-3039 (1991); Siegfried, G., et al., Endocrinology 136(3): 1267-1275 (1995)) though not in others (Reid, I. R., et al., Am. J. Physiol. 253(1 Pt 1):E45-51 (1987); Tamura, T., et al., Biochem. Biophys., Res. Commun. 159:1352-1358 (1989)). Studies of the signaling properties of the cloned type I PTHR have focused almost exclusively upon activation of AC, PLC or Cai++ (Abou-Samra, A. B., et al., Proc. Natl. Acad. Sci. U.S.A. 89(7):2732-2736 (1992); Bringhurst, F. R., et al., Endocrinology 132(5):2090-2098 (1993); Guo, J., et al., Endocrinology 136(9):3884-3891 (1995); Pines, M., et al., Bone 18(4):381-389 (1996); Jobert, A.-S., et al., Endocrinology 138(12):5282-5292 (1997); Schneider, H., et al., Eur. J. Pharm. 246(2):149-155 (1993)), although stimulation of PKC and of PKC-dependent ion transport by hPTH(1-34), hPTH-(3-34) and other hPTH fragments was reported in CHO cells transfected with rat PTHR cDNA (Azarani, A., et al., J. Biol. Chem. 271(25):14931-14936 (1996)).
Collectively, these observations have engendered the concept that the structural determinants for activation of AC/PKA signaling are distinct from those required for activation of PLC or PKC and that these reside, respectively, within the N- and C-terminal domains of PTH-(1-34) (Jouishomme, H., et al., J. Bone Miner. Res. 9(6):943-949 (1994); Tregear, G. W., and Potts, J. T., Jr. Endocr. Res. Commun. 2:561-567 (1975); Whitfield, J. F., and Morley, P. Trends Pharm. Sci. 16(11):382-386(1995)). In particular, the region hPTH-(29-32) was identified specifically as a critical PKC activation domain (Jouishomme, H., et al., J. Bone Miner. Res. 9(6):943-949 (1994); Whitfield, J. F., and Morley, P. Trends Pharm. Sci. 16(11):382-386 (1995)).
Compared with what is known from these studies of the rat PTHR, much less information is available regarding the structural features of human PTH required for binding to the human PTHR or for activation of its various signaling modes. Alanine-scanning mutagenesis has highlighted the importance of the C-terminal portion of hPTH-(1-34) for binding to the rat PTHR (30). Functional studies of transfected human PTHRs in COS-7 or HEK 293 cells have confirmed that hPTH-(1-34) activates AC and Cai++, although stimulation of PLC was not observed consistently and responses that were reported were modest (Pines, M., et al., Bone 18(4):381-389 (1996); Seuwen, K., et al., Brit. J. Pharm. 114(8):1613-1620 (1995); Jobert, A.-S., et al., Endocrinology 138(12):5282-5292 (1997); Schneider, H., et al., FEBS Lett. 351(2):281-285 (1994);). The effects of hPTH-(3-34) on Cai++ are similarly controversial (Pines, M., et al., Bone 18(4):381-389 (1996); Jobert, A.-S., et al., Endocrinology 138(12):5282-5292 (1997)), while the roles of other regions of the hPTH-(1-34) molecule in signaling via the human PTHR have not been systematically addressed. Synthetic hPTH-(1-30)NH2, hPTH-(1-29)NH2, hPTH-(1-28)NH2 hPTH-(1-27)NH2, and hPTH-(1-26)NH2 were each incapable of stimulating the activity of membrane-bound PKCs in osteoblast-like ROS17/2 cells (Neugebauer et al. (Biochem 34: 8835-8842 (1995)).
The relatively large size of native PTH presents challenges to the use of these peptides as treatments for osteoporosis. In general, a protein of this size is not suitable for use as a drug, since it cannot be delivered effectively by simple methods such as nasal inhalation. Instead, injection is required, and in the case of PTH, daily, or almost daily injections would most likely be needed to achieve increases in bone formation rates. Additionally, larger peptides are technically difficult and expensive to prepare by conventional synthetic chemistry methods. Alternative methods employing recombinant DNA and cell-based expression systems are also expensive, potentially vulnerable to contamination by foreign proteins and do not circumvent the delivery problem.
Accordingly, it would be advantageous for those skilled in the art to be able to identify a small molecule analog (either peptide or non-peptide) that is based on the larger peptide and yet which still retains the desired biological activities. The activity is greater relative to the intact peptide, but further optimization can lead to enhanced efficacy and potency.
We recently observed that hPTH-(1-31), shown by others to be a full AC agonist but to be incapable of activating PKC via rodent PTHRs (Jouishomme, H., et al., Endocrinology 130(1):53-60 (1992)), was as potent as hPTH-(1-34) in activating both AC and PLC via human PTHRs expressed in LLC-PK1, COS-7 or HEK 293 cells (Takasu, H., and Bringhurst, F. R., Endocrinology 139(10):4293-4299 (1998)). These unexpected observations prompted us to undertake a more detailed analysis of the relative roles of the extreme N-terminal region of hPTH-(1-34) in binding to, and selective activation of the human PTH-1/PTH-2 receptor, with a particular focus on PLC activation.
The present invention is directed to amino terminal modifications of human parathyroid hormone 1-34 peptide which selectively activate the PTH-2 receptor much more effectively than does the native hPTH(1-34)NH2 ligand but which still activate the classical, PTH-1 receptor, albeit at a reduced level. This unexpected finding has important implications for the design of excellent receptor selective PTH agonists in systems including in vivo where both receptors are expressed. Importantly, the desamino peptides of the present invention are still signal selective via the PTH-1 receptor in that they possess some cAMP activity but minimal PLC activity. Accordingly, the desamino peptides described herein will be useful as bone anabolic agents for the therapeutic treatment of bone disease in vivo.
The present invention relates to PTH(1-34) peptides and derivatives thereof. Compounds of the invention which include PTH(1-34) peptides, fragments thereof, derivatives thereof, pharmaceutically acceptable salts thereof, and Nxe2x80x94 or Cxe2x80x94 derivatives thereof, are hereinafter collectively referred to as xe2x80x9ccompounds of SEQ ID NO:1 and derivatives thereof.xe2x80x9d
The invention provides synthetic and/or recombinant biologically active peptide derivatives of PTH(1-34). In one specific embodiment, this invention provides a biologically active peptide at least 90% identical to a peptide consisting essentially of the formula:
(a) X01 ValSerGluIleGlnLeuMetHisAsnLeuGlyLysHisLeuAsnSerMet GluArgValGluTrpLeuArgLysLysLeuGlnAspValHisAsnPhe(SEQ ID NO:1);
(b) fragments thereof containing amino acids 1-29, 1-30, 1-31, 1-32, or 1-33;
(c) pharmaceutically acceptable salts thereof; or
(d) Nxe2x80x94 or Cxe2x80x94 derivatives thereof,
xe2x80x83wherein:
X01 is desamino Ser, desamino Ala or desamino Gly,
provided that said peptide is not desamino Ser1 hPTH(1-31)NH2 or desamino Ser1 hPTH(1-34)NH2.
In accordance with yet a further aspect, this invention also provides pharmaceutical compositions comprising a biologically active peptide at least 90% identical to a peptide consisting essentially of the formula:
(a) X01 ValSerGluIleGlnLeuMetHisAsnLeuGlyLysHisLeuAsnSerMet GluArgValGluTrpLeuArgLysLysLeuGlnAspValHisAsnPhe(SEQ ID NO:1);
(b) fragments thereof containing amino acids 1-29, 1-30, 1-31, 1-32, or 1-33;
(c) pharmaceutically acceptable salts thereof; or
(d) Nxe2x80x94 or Cxe2x80x94 derivatives thereof;
xe2x80x83wherein:
X01 is desamino Ser, desamino Ala or desamino Gly; and a pharmaceutically acceptable carrier.
In accordance with yet a further aspect, this invention provides a nucleic acid molecule consisting essentially of a polynucleotide encoding a biologically active peptide which has an amino acid sequence selected from the group consisting of:
(a) X01 ValSerGluIleGlnLeuMetHisAsnLeuGlyLysHisLeuAsn SerMetGluArgValGluTrpLeuArgLysLysLeuGlnAspValHisAsnPhe (SEQ ID NO:1);
(b) fragments thereof containing amino acids 1-29, 1-30, 1-31, 1-32, or 1-33;
xe2x80x83wherein:
X01 is desamino Ser, desamino Ala or desamino Gly,
provided that said peptide is not desamino Ser1 hPTH(1-31)NH2 or desamino Ser1 hPTH(1-34)NH2.
In accordance with yet a further aspect, this invention provides a recombinant DNA molecule comprising: (1) an expression control region, said region in operable linkage with (2) a polynucleotide sequence coding for a biologically active peptide, wherein said peptide is selected from the group consisting of:
(a) X01 ValSerGluIleGlnLeuMetHisAsnLeuGlyLysHisLeuAsn SerMetGluArgValGluTrpLeuArgLysLysLeuGlnAspValHisAsnPhe (SEQ ID NO:1);
(b) fragments thereof containing amino acids 1-29, 1-30, 1-31, 1-32, or 1-33;
xe2x80x83wherein:
X01 is desamino Ser, desamino Ala or desamino Gly, provided that said peptide is not desamino Ser1 hPTH(1-31)NH2 or desamino Ser1 hPTH(1-34)NH2.
In accordance with yet a further aspect, this invention provides a method for treating mammalian conditions characterized by decreases in bone mass, which method comprises administering to a subject in need thereof an effective bone mass-increasing amount of a biologically active peptide, wherein said peptide comprises an amino acid sequence at least 90% identical to a member selected from the group consisting essentially of:
(a) X01 ValSerGluIleGlnLeuMetHisAsnLeuGlyLysHisLeuAsn SerMetGluArgValGluTrpLeuArgLysLysLeuGlnAspValHisAsnPhe (SEQ ID NO:1);
(b) fragments thereof containing amino acids 1-29, 1-30, 1-31, 1-32, or 1-33;
(c) pharmaceutically acceptable salts thereof; or
(d) Nxe2x80x94 or Cxe2x80x94 derivatives thereof;
xe2x80x83wherein:
X01 is desamino Ser, desamino Ala or desamino Gly,
provided that said peptide is not desamino Ser1 hPTH(1-31)NH2 or desamino Ser1 hPTH(1-34)NH2; and a pharmaceutically acceptable carrier.
In accordance with yet a further aspect, there is provided a method for treating a medical disorder that results from altered or excessive action of the PTH-2 receptor, comprising administering to a patient a therapeutically effective amount of a biologically active peptide wherein said peptide comprises an amino acid sequence at least 90% identical to a member selected from the group consisting essentially of:
(a) X01 ValSerGluIleGlnLeuMetHisAsnLeuGlyLysHisLeuAsn SerMetGluArgValGluTrpLeuArgLysLysLeuGinAspValHisAsnPhe (SEQ ID NO:1);
(b) fragments thereof containing amino acids 1-29, 1-30, 1-31, 1-32, or 1-33;
(c) pharmaceutically acceptable salts thereof, or
(d) Nxe2x80x94 or Cxe2x80x94 derivatives thereof;
xe2x80x83wherein:
X01 is desamino Ser, desamino Ala or desamino Gly,
provided that said peptide is not desamino Ser1 hPTH(1-31)NH2 or desamino Ser1 hPTH(1-34)NH2; and a pharmaceutically acceptable carrier sufficient to activate the PTH-2 receptor but not the PTH-1 of said patient.
In accordance with yet a further aspect, this invention also provides a method for determining rates of bone reformation, bone resorption and/or bone remodeling comprising administering to a patient an effective amount of a labeled peptide of SEQ ID NO: 1 or a derivative thereof and determining the uptake of said peptide into the bone of said patient. The peptide may be labeled with a label selected from the group consisting of: radiolabel, flourescent label, bioluminescent label, or chemiluminescent label. An example of a suitable radiolabel is 99m Tc.
In accordance with yet a further aspect of the invention, any amino-acid substitutions at positions 1-34, and more particularly those amino acid substitutions at amino acid position 1, which do not destroy the biological activity of the PTH(1-34) peptide analog to selectively agonize the PTH-1/PTH-2 receptor (as determined by assays known to the skilled artisan and discussed below), are also included within the scope of the present invention.